PROJECT SUMMARY Cancers evolve through the sequential acquisition of mutations that collectively perturb cellular homeostasis thereby unleashing havoc. How do these deranged cells survive, form tumors, amass additional deleterious mutations and acquire resistance to therapies? I propose to tackle these important questions using a novel approach founded on principles of protein folding. My work has demonstrated that the protein-folding chaperone heat-shock protein 90 (HSP90) alleviates or ?buffers? the deleterious biological effects of human mutations. In doing so, HSP90 alters cellular drug sensitivities and influences the clinical course of diverse Mendelian disorders. Here, I hypothesize that by buffering mutations, HSP90 enables tumor cells to thwart cancer therapies. The proposed work will determine how HSP90, acting epigenetically, alters the consequences of specific DNA repair mutations in cancers, and how HSP90 buffering influences tumor responses to genotoxic therapies. I will directly address these questions by measuring the effects of malignant transformation itself on HSP90's ability to buffer mutations in DNA repair proteins. I will determine the ability of HSP90 to buffer a panel of well-characterized mutants in the Fanconi Anemia (FA) DNA repair pathway, using cell lines transformed in vitro by transduction with defined oncogenic elements, and using genetically engineered FA patient-derived cancer cell lines. As a therapeutically relevant complement to these studies, I will also investigate the role of HSP90 in buffering mutant DNA repair proteins and how HSP90 helps shape tumor responses to genotoxic drugs. I will employ a high-throughput functional genetics approach to parse clinical cancers based on the presence of HSP90-buffered vs. non-buffered mutations (defined by my recently published and ongoing work) in DNA repair proteins. Results will be correlated with clinical outcome following standard-of-care treatment with genotoxic drugs. This K22 award will help me secure the funds for an independent position at a leading institution, establish a record of independent research, and generate preliminary data for the R01 phase of my career. It will also provide me with training in cancer biology and patient-oriented research skillsets that will help me compete for a broader spectrum of funding opportunities. The work I propose will determine how the influence of HSP90 on the accumulation of mutations evolves as cancers progress and how its evolving role enables the emergence of drug resistance. Results will provide a theoretical framework permitting the identification and use of HSP90-buffered mutations for patient stratification. This will contribute to the design of individualized treatments likely to provide the most benefit, including the use of existing HSP90 inhibitors in non-conventional ways. Thus, this work will provide important insights into fundamental mechanisms of tumor evolution as well as pioneer a strategy for improving the precision and efficacy of therapeutic interventions relevant to the treatment of many cancers.